Implantable medical devices having double walled microspheres

ABSTRACT

An implantable medical device including at least one double-walled microsphere containing an active agent, and a biodegradable polymer layer containing the at least one double-walled microsphere.

PRIORITY CLAIM

This application is a divisional application of U.S. patent applicationSer. No. 15/334,625 filed Oct. 26, 2016, which is a divisionalapplication of U.S. patent application Ser. No. 13/788,879 filed Mar. 7,2013 (now U.S. Pat. No. 9,498,221), which is a continuation ofInternational Application Serial No. PCT/US2011/051053 filed Sep. 9,2011, which claims priority to U.S. Provisional Application Ser. No.61/381,725 filed Sep. 10, 2010, to each of which priority is claimed andthe contents of each of which are incorporated by reference herein intheir entireties.

GRANT INFORMATION

This invention was made with government support under grants DMR-0705948awarded by the National Science Foundation and W81XWH-07-1-0716, awardedby the Department of Defense. The government has certain rights in theinvention.

1. INTRODUCTION

The present invention relates to implantable medical devices (e.g.,nerve guides) having double walled microspheres.

2. BACKGROUND

Though there has been a considerable amount of research in improvingperipheral nerve guide design, commercially available nerve guides havenot equaled the regenerative capacity of the nerve autograft in long gapperipheral nerve repair. While autografts (e.g., the sural nerve) havebeen used to bridge nerve defects of 6 cm or more (Kim et al., 2008,“Nerve Injuries:operative results from major nerve injuries,entrapments, and tumors. 2nd ed. Philadelphia:Saunders Elsevier. pp.1-611), polymer based nerve guides are effectively used to regeneratenerves in gaps that span only 3 cm or less (Schlosshauer et al., 2006,Neurosurgery 59(4):740-748). This barrier in gap length may reflect theunmet need for nerve guides to actively promote nerve regenerationthrough the lumen of the guide from the proximal to the distal nervestump (Kemp et al., 2008, Neurol. Res. 30:1030-1038).

In addition to providing mechanical support for regenerating nerves,nerve guides should also provide cues that guide axonal growth andincrease the rate at which nerves regenerate.

The complex problem of targeting axonal outgrowth has led to theinvestigation of a variety of nerve guide materials, luminal fillers,cell therapies and combinations thereof (Midha et al., 2003, J.Neurosurg. 99(3):555-565). One additional pathway toward enhancingaxonal growth involves locally delivering drugs or neurotrophic factorsthat promote nerve growth and survival. Growth factors have beendelivered from nerve guides by adsorbing the growth factor to the nerveguide scaffold, incorporating growth factors into the scaffold materialduring fabrication (Chavez-Delgada et al., 2003, J. Biomed. Mater. Res.B. Appl. Biomater. 67B(2):702-711; Yang et al., 2005, J. Control Release104(3):433-446), embedding growth factor loaded rods or microspheresinto the nerve guide (Fine et al., 2002, Eur. J. Neurosci.15(4):589-601; Bloch et al., 2001, Exp. Neurol. 172(2):425-432; Xu etal., 2003, Biomaterials 24(13):2405-2412; Rosner et al., 2003, Ann.Biomed. Eng. 31(11):1383-1401; Goraltchouk et al., 2006, J. ControlRelease 110(2):400-407; Singh et al., 2008, Tissue Eng Part C Methods14(4):299-309; Dodla et al., 2008, Biomaterials 29(1):33-46), covalentlyimmobilizing growth factors onto the nerve guide surface (Chen et al.,2006, J. Biomed. Mater. Res. A. 79A(4):846-857; Wood et al., 2009, J.Biomed. Mater Res A 89A(4):909-918; Lee et al., 2003, Exp. Neurol.184(1):295-303), or by implantation of an osmotic pump (Newman et al.,1996, Arch Otolaryngol Head Neck Surg 122(4):399-403; Lewin et al.,1997, Laryngoscope 107(7):992-999) (for review of these techniques, seeKemp et al., 2008, Neurol. Res. 30:1030-1038 and Willerth et al., 2007,Adv. Drug Deliv. Rev. 59(4-5):325-338). Results from these preclinicalstudies have shown beneficial effects of delivered growth factors fornerve regeneration. For example, the delivery of nerve growth factor(NGF) promotes sensory neuron survival, outgrowth and branching (Blochet al., 2001, Exp. Neurol. 172(2):425-432), ciliary neurotrophic factor(CNTF) aids in motor neuron survival and outgrowth (Xu et al., 2009, J.Clin. Neurosci. 16(6):812-817) and glial cell line-derived neurotrophic(GDNF) has been shown to promote the regeneration of fibers originatingfrom the spinal cord beyond what has been measured with NGF (Fine etal., 2002, Eur. J. Neurosci. 15(4):589-601). Because of the promisingreports following treatment of nerve injuries with neurotrophic factors,the delivery of such factors can be a potential method of surpassing thecurrent length limitations in nerve regeneration. While strategies havebeen developed for protein delivery from polymer nerve guides, manyconduit delivery systems lack a sustained and controlled release rate ofbioactive proteins for the entire duration required for the axon tocross from the proximal to the distal nerve stump. Accordingly, thereremains a need for nerve guides and other medical devices that candeliver an active agent for therapeutically effective periods.

3. SUMMARY

One embodiment of the presently disclosed subject matter provides animplantable medical device that includes double-walled microspherescontaining an active agent, and a biodegradable polymer containing thedouble-walled microsphere. In one embodiment, the double-walledmicrosphere can include a poly(lactide) wall and apoly(lactic-co-glycolic acid) wall, where the active agent is aneurotrophic factor, such as glial cell-line derived neurotrophic factor(GDNF) or glial growth factor 2 (GGF2). The biodegradable polymer layercan include poly(caprolactone), and double walled microspheres can beembedded in the biodegradable polymer layer. A second biodegradablepolymer can further be applied to the biodegradable polymer layercontaining the at least one double-walled microsphere, and, withoutlimitation can encapsulate the first, microsphere-containing polymer. Inone embodiment, the medical device is a nerve guide.

Another embodiment of the presently disclosed subject matter provides amethod of making an implantable medical device that includes forming afirst mixture (e.g., a solution) of a first polymer and a first solvent,forming a second mixture (e.g., a solution) of a second polymer and asecond solvent, adding an active agent to the first mixture and/or thesecond mixture, combining the first mixture and the second mixture toform a third mixture, introducing the third mixture to a third solventto form microspheres, isolating the microspheres from the third solvent,and introducing the isolated microspheres into a biodegradable polymerlayer. In one embodiment, the first mixture and the second mixture arecombined by vortexing to form the third mixture, and the third mixtureis added dropwise to the third solvent. In one embodiment, an emulsionis formed upon combining the first and second mixture. The microspherescan be isolated from the third solvent by centrifugation.

In one embodiment, the first polymer is poly(lactic-co-glycolic acid)and/or the second polymer is poly(lactide) (e.g. poly(L-Lactide)). Theactive agent is a neurotrophic factor, such as GDNF or GGF2.

Another embodiment of the present application provides an implantablenerve guide that includes double-walled microspheres with a neurotrophicfactor active agent encapsulated therein, the double walled comprised ofa poly(lactide) layer (“wall”) and a poly(lactic-co-glycolic acid) layer(“wall”), a poly(caprolactone) polymer layer containing thedouble-walled microspheres; and a second poly(caprolactone) layer thatat least partially encapsulates the biodegradable polymer layercontaining the double-walled microspheres. In one embodiment, the nerveguide releases GDNF or GGF2 for at least 50 days following implantationin a human.

Another embodiment of the present application provides a method ofpromoting nerve generation comprising implanting an implantable medicaldevice or an implantable nerve guide, as those medical devices and nerveguides are described herein. Yet another embodiment provides animplantable medical device or an implantable nerve guide prepared by anyone of the processes described herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a is a photograph of PCL disks following fabrication in custommade silicone mold.

FIG. 1b is a schematic of polymer orientation in double-walledmicrosphere.

FIG. 1c is a schematic of a fabrication technique for incorporatingdouble-walled microspheres into PCL nerve guide.

FIG. 2a is a photograph of PCL guide at time of implantation. Rulerindicates nerve guide is 1.7 cm in length. Nerve stumps were inserted 1mm into each end of nerve guide.

FIG. 2b is a photograph of explanted nerve and conduit at 6 weeks. Guidehas been fixed and treated with osmium tetroxide. Dashed likes indicateposition on guide where transverse cuts were made for histology.

FIG. 2c is a photograph of sectioned nerve guide embedded in paraffin.

FIG. 3a depicts the cumulative release of GDNF from double-walledmicrospheres (mean±stdev, n=4 for 1 batch).

FIG. 3b depicts the weight of nerve guides following NaCl impregnationand removal by leaching in distilled water for specified time points.(Mean±stdev, N=5).

FIG. 3c depicts the cumulative release of the GDNF (pg) per individualnerve guide (1.7 cm in length), with values expressed as mean±std. dev.(n=4).

FIG. 4a is a compilation of brightfield and fluorescent micrographstaken at 10×. Multiple images of both the nerve guide and FITC-BSAencapsulated in double-walled microspheres were used to overlayfluorescent images on brightfield prior to realignment of figures.

FIG. 4b is a scanning electron micrograph of double-walled microspherefollowing incorporation into PCL nerve guides.

FIG. 5a depicts the cumulative release of lysozyme from PCL disks (A,diamonds) and microspheres suspended in buffer solution (B, squares)(mean±stdev, N=5). Asterisks indicate statistical differences betweenmicrospheres that were suspended in solution and those embedded in disks(p<0.05).

FIG. 5b depicts the cumulative release of lysozyme from nerved guideswith double-walled microspheres (mean±stdev, n=5).

FIGS. 6a and 6b are a compilation of brighfield micrographs taken of atransverse section of the proximal segment guides visualized withMasson's trichrome stain. To create the final image, a series of 10×images were realigned and blended using photoshop CS3. FIG. 6A depictsnegative control guides. Microspheres are labeled as “MS.” The collagencapsule surrounding the implanted guide is labeled with an arrow. FIG.6B depicts guides releasing GDNF.

FIGS. 6c and 6d are high magnification brightfield micrographs taken ofthe lumen of explanted nerve guides from negative controls (FIG. 6c )and guides releasing GDNF (FIG. 6d ). Blood vessels are labeled with anarrow head. Scale bars are 100×.

FIGS. 7a and 7b are compilation of brighfield micrographs taken of atransverse section of the distal segment of guides visualized withMasson's trichrome stain. FIG. 7A depicts the negative control guidesand FIG. 7b depicts guides with GDNF.

FIGS. 7c and 7d depict high magnification brightfield micrographs takenof the lumen of explanted nerve guides from negative controls (FIG. 7c )and guides releasing GDNF (FIG. 7d ). Scale bars are 100 μm.

FIGS. 8a and 8b are black and white fluorescent micrograph showingSchwann cells (visualized with S100). Scale bars are 100 μm. FIG. 8adepicts the proximal transverse section of negative control guides. FIG.8b depicts the proximal section of guides release GDNF.

FIG. 8c depicts high magnification fluorescent micrograph of negativecontrol nerve guide. FIG. 8d depicts high magnification fluorescentmicrograph of double-walled microsphere encapsulating GDNF. Redindicates S100 labeling of Schwann cells. Dapi is blue.

FIGS. 9a and 9b are fluorescent micrographs from distal segment ofexplanted nerve tissue for a negative control (FIG. 9a ) and a guideimplanted with encapsulated GDNF (FIG. 9b ). Red indicates S100 proteinin Schwann cells and Blue is DAPI nuclei stain.

FIG. 10a shows a photo of high-speed camera with equipment for dataacquisition used for recording videos of animal gait.

FIG. 10b shows a photo of anatomical placement of reflective markers forcalculating joint angles during gait.

FIG. 10c shows a photo of a rat traversing walkway while being videorecorded for gait analysis.

FIG. 11 shows an image of a rat captured during plantarflexionimmediately before initiating the swing phase. Line segments connecteach of the five reflective markers. Representative vectors indicatedwith A and B are used to calculate the intersegmental joint angle y.Color images available online at www.liebertonline.com/tea.

FIG. 12a shows a photo and FIG. 12b shows a schematic of a rat preparedon a data acquisition board for muscle contraction force recordings.Color images available online at www.liebertonline.com/tea.

FIG. 13a shows a photo of exposed empty PCL nerve guide 16 weeks afterimplantation (white arrow). The proximal and distal ends of the guideare indicated with the characters P and D.

FIG. 13b shows a photo of GDNF releasing PCL nerve guide (white arrow)that has been longitudinally sectioned revealing regenerated nerve(white arrowhead). Color images available online atwww.liebertonline.com/tea.

FIG. 14A-F shows hip range of motion (degrees) at baseline (week 0) andsequential timepoints after injury for swing phase (A) and stance phase(B) of the gait cycle. Knee range of motion during swing (C) and stance(D). Ankle range of motion (degrees) in swing (E) and stance (F). Forall graphs, GDNF animals are represented with (♦), control PCL guidesare (▪) and isografts at week 15 are (▴).

FIG. 15 shows the average gastrocnemius contraction force (N) with errorbars representing standard deviation. *=p<0.05.

FIG. 16A-D shows transverse sections of the proximal segment of guidesexplanted after 16 weeks as observed with Masson's trichrome stain. (A)Low-magnification brightfield micrographs taken of negative controlguides with regenerated nerve tissue indicated within black box. (B)High magnification micrographs showing detailed nerve tissueorganization. (C) Low magnification transverse section from guidesreleasing GDNF with centrally located nerve tissue circled in black. (D)High magnification brightfield image of nerve fiber organization afterGDNF treatment. Scale bars are 100 mm. NG ¼ nerve guide. Color imagesavailable online at www.liebertonline.com/tea.

FIG. 17A-F shows transverse sections from guides explanted 16 weeks postinjury observed with Masson's trichrome stain. Lowmagnificationbrightfield micrographs taken of negative control guides from the mid(A) and distal (B) sections of the explanted guide. (C)Low-magnification mid-transverse section from guides releasing. (D)High-magnification brightfield image of nerve fiber organization afterGDNF treatment. (E) Low-magnification distal transverse section fromguides releasing. (F) High-magnification brightfield image showingdetail nerve fiber organization from within GDNF guides. Scale bars are100 mm. Color images available online at www.liebertonline.com/tea.

FIG. 18A-F Fluorescent micrographs of proximal transverse sections ofnegative control guides (A) and guides releasing GDNF (B). Fluorescentmicrograph from middle segment of control (C) and GDNF (D) nerve guides.Distal transverse section from negative control (E) and GDNF releasingnerve guides (F). Red indicates S100 labeling of Schwann cells.Neurofilament proteins within nerve fibers are green.40,6-Diamidino-2-phenylindole is blue. Scale bars are 100 mm. Colorimages available online at www.liebertonline.com/tea.

FIG. 19A-B. (A) shows calculated g-ratio of fibers within transversesegments of the PNS, PG, DG, and DNS. (B) Calculated nerve fiber density(fibers per mm2). Treatment groups are isograft (▪), GDNF nerve guide(□) and control nerve guide (□). Values are expressed as mean|standarddeviation. Gray horizontal bar indicates normal, uninjured g-ratiovalues. PNS, proximal nerve stump; PG, proximal graft or guide; DG,distal graft or guide; DNS, distal nerve stump. *=p<0.05.

5. DETAILED DESCRIPTION

A biodegradable polymer medical device having double-walled microspheresis provided that locally delivers an active agent (e.g., bioactiveneurotrophic factor) in physiologically relevant concentrations forpre-selected periods (e.g. for at least 50 days). While the presentlydisclosed subject matter will be, for convenience, largely discussedwith reference to a nerve guide, the presently disclosed subject matteris equally applicable to any medical device for which it is desired todeliver any active agent over an extended period of time. The deviceand/or microspheres of the invention may be used in a human, non-humanprimate, non-human mammal, rodent, or other non-human animal subject.

One embodiment of the presently disclosed subject matter provides abiodegradable polymer nerve guide that allows transected peripheralnerves to cross from a proximal to a distal nerve stump. Delivery of aneurotrophic factor enhances regeneration and overcomes currentlimitations in nerve repair across large defects. Glial CellLine-Derived Neurotrophic Factor (GDNF) is a promoter of axonalelongation and branching and has shown promising pre-clinical results inanalysis of nerve regeneration with nerve guides. In addition, GDNF hasbeen shown to promote Schwann cell proliferation and migration. Othernerve factors that may be comprised in the microspheres include, but arenot limited to, glial growth factor 2 (GGF2), brain-derived neurotrophicfactor (BDNF), novel neurotrophin-1 (NNT1), Ciliary neurotrophic factor(CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). Theseagents, or a combination thereof, can be provided in addition to, or inplace of, GDNF.

While the application has been described, solely for convenience, in thecontext of a nerve guide that employs a neurotrophic factor, the presentinvention encompasses other medical devices having double-walledmicrospheres containing active agent(s), where said microspheres arecontained in a biodegradable polymer. Selection of the active agent canbe made based on the function of the medical device, and thephysiological needs of the subject to be treated. For example, and notby way of limitation, other agents that can be incorporated intodouble-walled microspheres include chemotherapeutic drugs (e.g.,doxorubicin and/or cisplatin), insulin, dexamethasone, bone morphogenicprotein-2, transforming growth factor β1, fibroblast growth factors 1and 2, antihyperglycemic drugs (e.g., pioglitazone), kinase inhibitors,and combinations thereof.

Non-limiting examples of kinase inhibitors that may be incorporated intodouble-walled microspheres include glycogen synthase kinase 3 (GSK3)inhibitors such as SB-415286, LiCl, insulin-like growth factor-1, orSB216763, and agents set forth in: Cohen and Goedert, 2004, Nat. Rev.Drug Disc. 3(6): 479-487; Bhat et al., 2004, J. Neurochem. 89(6):1313-1317; and MacAubay et al., 2003, Eur. J. Biochem. 270(18):3829-3838; Rho/ROCK inhibitors such as fasudil or Y-27632 and agents setforth in Park, et al., 2011, J. Pharmacol. Exp. Ther. 338(1): 271-279;and Micuda et al., 2010, Curr. Cancer Drug Targets 10(2): 127-134; andJNK inhibitors such as SP600125 and “bidentate molecule 19” and agentsset forth in Bogoyevitch and Arthur, 2008, Biochim. Biphys. Acta1784(1): 76-93; Manning and Davis, 2003, Nat. Rev. Drug Discov. 2(7):554-565; Stebbins et al., 2011, J. Med. Chem. PMID=21815634; and Boweset al., 2011, Bioorg. Med. Chem. Lett. 21(18): 5521-5527.

In one embodiment, summarized in greater detail in the Examples below, adouble-walled microsphere delivery system is provided for delivery of anactive agent (e.g., bioactive GDNF) with a sustained release profile ofat least 7 days, or at least 14 days, or in one preferred embodiment, atleast 50 days. In this particular embodiment, microspheres, preferablydouble-walled microspheres, are incorporated within a degradablepoly(caprolactone) nerve guide in a reproducible distribution.Implantation of nerve guides across a 1.5 cm defect in a rat sciaticnerve gap resulted in an increase in tissue integration in both theproximal and distal segments of the lumen of the nerve guide after 6weeks. In addition, transverse sections of the distal region of theexplanted guides showed the presence of Schwann cells while none weredetectable in negative control guides. Migration of Schwann cells todouble-walled microspheres indicated that bioactive GDNF wasencapsulated and delivered to the internal environment of the nerveguide. Because GDNF increased tissue formation within the nerve guidelumen and also promoted the migration and proliferation of Schwanncells, the presently disclosed nerve guides can promote nerveregeneration beyond that capable with pre-existing nerve guides.

In one embodiment, double-walled microspheres including poly(lactide)and poly(lactic-co-glycolic acid) walls are incorporated into porouspoly(caprolactone) nerve guides. The poly(lactide) wall can be the innerwall and the poly(lactic-co-glycolic acid) wall can be outer wall.Alternatively, the poly(lactide) wall can be the outer wall and thepoly(lactic-co-glycolic acid) wall can be inner wall.

The order of the walls (that is to say, which polymer becomes the innerwall and which polymer becomes the outer wall) can be determined basedon the principles of phase separation. For example, once solutionscontaining the two polymer “walls” are mixed to form an emulsion, thepolymer layer that is first to precipitate out the solvent associatedtherewith (i.e. the solvent that is first to evaporate) will form thecore layer, and the later-precipitating polymer will form the shell.Persons of ordinary skill in the art can obtain the desired wall orderbased on, for example, the hydrophilicity of the solvent selected, thepolarity of the solvent selected, and the solubility profile of thepolymer itself. Phase separation techniques are known to those ofordinary skill in the art, and details can be found, for example, in “Invitro and in vivo degradation of double-walled polymer microspheres,”Journal of Controlled Release 40:169-178 (1996), and “In vitrodegradation of polyanhydride/polyester core-shell double-walledmicrospheres,” International Journal of Pharmaceutics, 301:294-303(2005), each of which is hereby incorporated by reference in theirentirety.

Bioactive proteins can be released from double-walled microspheres forover 80 days [21]. Double-walled microspheres can be reproduciblyintegrated within polymer nerve guides in manufacturer-controlleddistribution. To confirm the distribution of microspheres within thenerve guide, fluorescently labeled bovine serum albumin (BSA) wasencapsulated and visualized through fluorescent microscopy. Becauseparticulate leaching is required to create the porous nerve guidewalls,the minimum period required for sodium chloride removal was determined.In vitro release studies were performed to determine the effect of thenerve guide macrostructure on release of a model protein, lysozyme. Theoverall efficacy of this fabrication technique was confirmed with GDNFin the rat sciatic nerve model. For evaluating the feasibility of thenerve guide design, a time period of only 6 weeks was used within theanimal model. The rationale for this early time point was to firstestablish the bioactivity of proteins delivered within the lumen of thenerve guide from microspheres embedded within the nerve guide wall.

Results show that tissue integration within GDNF releasing nerve guideswas improved with a greater concentration of intercellular fibers andcollagen content. Furthermore, a localization of Schwann cells aroundmicrospheres encapsulating GDNF indicates that bioactive GDNF was beingreleased from our delivery system.

While initial studies for evaluating nerve repair were conducted with asingle growth factor, this technique could lend itself useful forevaluating delivery systems for additional growth factors orcombinations thereof.

The presently disclosed subject matter also provides methods of makingan implantable medical device (e.g., a nerve guide). In one embodiment,the method includes forming a first mixture (e.g., a solution) of afirst polymer and a first solvent, and separately, forming a mixture(e.g., a solution) of a second polymer and a second solvent. The firstor second polymer, which will ultimately form a wall of the doublewalled microsphere can be, for example, poly(l-lactide),poly(lactic-co-glycolic acid), poly(1,3-bis-(p-carboxyphenoxypropane)-co-(sebacic anhydride) (e.g., 20:80 PCPP:SA),poly(fumaric-co-sebacic) anhydride, and poly[(1,6-bis-carboxyphenoxy)hexane]. The first or second solvent can be, for example, waterdichloromethane, ethyl acetate, diethyl ether, THF, acetone and EMSO.

For example, in an alternative embodiment, polylactic acid) can form theexternal layer and poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacicanhydride) (e.g., 20:80 PCPP:SA) can form the core layer.

After a polymer is added to the mixture, and preferably after thepolymer is fully dissolved, an active agent (e.g., GDNF) is then addedto the first and/or second mixture. Alternatively, a component that isreadily discernable (e.g., fluorescently-labeled bovine serum albumin)can be added in place of, or in addition to, the active agent forpurposes of testing. Examples of active agents that can be added to thefirst and/or second mixture, and ultimately the medical device itselfinclude neurotrophic factors, such as, but not limited to, glialcell-line derived neurotrophic factor (GDNF), brain-derived neurotrophicfactor (BDNF), novel neurotrophin-1 (NNT1), Ciliary neurotrophic factor(CNTF), and neurotrophin-3 (NT-3). After adding the active agent, themixture can be vortexed for a period of time (e.g., less than a minute)to achieve a homogenous mixture. An emulsifier such as docusate sodiumsalt can also be added to the first and/or second mixtures. Anemulsifier, such as docusate sodium, or other stabilizer can be added tothe first and/or second mixtures to stabilize the protein.

The two mixtures can then be combined, and optionally vortexed. Whenpoly(lactide) and poly(lactic-co-glycolic acid) polymers are used aswall-forming polymers and dichloromethane is used as the solvent for thefirst two mixtures, an oil-in-oil emulsion is formed upon combining thetwo mixtures. The combined mixtures can be gradually added to a thirdsolvent (e.g., drop-wise using a Pasteur pipette) to form a thirdmixture and then vigorously stirred for a few hours (e.g., stirred at900 rpm for 3 hours). An aqueous solvent such as an aqueous solution of0.5% poly(vinyl alcohol) can be used as the third solvent.

When polymer mixtures are combined to form the third mixture (e.g., anemulsion), the polymer that is associated with the solvent that is thefirst to evaporate will form the core of the microsphere, and thepolymer that is associated with the solvent that is the last toevaporate will form the shell of the microsphere and at leastsubstantially encapsulate the core polymer wall. In embodiments in whichpoly(lactide) and poly(lactic-co-glycolic acid) polymers are used aswall-forming polymers and dichloromethane is used as the solvent for thefirst two mixtures, the poly(lactic-co-glycolic acid) polymer will formthe core layer, and the poly(lactide) will form the shell layer.

Microspheres are formed in the third mixture, which can be isolated, forexample, by centrifugation and washing. For example, the third mixturecan be centrifuged for about 10 minutes and washed with water, and thenrepeated. The microspheres obtained from the isolation step can then belyophilized and stored at a low temperature in a desiccant.

Medical devices to which the double-walled microspheres are added, suchas nerve guides can be prepared according to known methods. For example,double walled microspheres and nerve guides can be prepared as generallydisclosed in Kokai et al., Diffusion of soluble factors throughdegradable polymer nerve guides: controlling manufacturing parameters,Acta Biomater 2009; 5(7):2540-50, which is hereby incorporated byreference.

In one embodiment, polycaprolactone nerve guides can be prepared bycoating glass capillary mandrels with an aqueous polymer solution (e.g.,a 17% w/v aqueous solution of poly(vinyl alcohol)). The coated capillarymandrels can then be introduced to a polymer slurry of polycaprolactonedissolved in an organic solvent (e.g., ethyl acetate) to which sodiumchloride or other leaching salt has been added. The organic solvent isallowed to evaporate and the mandrel is again dipped in the polymerslurry. The resulting polymer conduits are immersed in distilled water,and the polymer subsequently removed from the glass mandrels. Thethickness of the polymer nerve guide can be varied based on the numberof immersions in the polymer slurry. For example, 6 immersions inpolymer slurry yielded a nerve guide wall thickness of about 600-700 μm.

The double-walled microspheres can be added to the medical device byintroducing the microspheres to the medical device in between immersionsin the polymer slurries. For example, the semi-dried, dip-coated medicaldevice can be introduced to microspheres that are even spread across anon-reactive surface (for example, but now by way of limitation,parchment paper) by rolling, or by similar means, and allowed to drybefore subsequent dip-coating. Any technique can be used to apply themicrospheres to the biodegradable, yet otherwise inert polymer (e.g.polycaprolactone) so long as the process does not dissolve or otherwisenegatively impact the microspheres. For example, processes requiringhigh temperatures should be disfavored, unless the microspheres can beprotected. Electrospinning and polymer casting techniques known to thoseof ordinary skill in the art can be used.

The present invention further provides for, but is not limited to, nerveguides prepared by the method set forth above.

6. EXAMPLE Incorporation of Double-Walled Microspheres Into PolymerNerve Guides 6.1 MATERIALS AND METHODS

Reagents: All chemicals were analytical grade or purer and werepurchased from commercial suppliers. Poly(vinyl alcohol) (average Mw9000-10,000, 80% hydrolyzed), poly(DL-lactide-co-glycolide)(lactide:glycolide (50:50), mol wt 40,000-75,000 units),poly(caprolactone) (Mw 65,000), Fluorescein isothiocyanate conjugatedBovine Albumin (A9771) (FITC-BSA), lysozyme from chicken egg white,dichloromethane, ethyl acetate, Gel Mount (G0918), xylene, monoclonalanti-S100, and Phosphate Buffered Saline (PBS) were all purchased fromSigma-Aldrich (St. Louis, Mo.). Poly-L-lactide (0.90-1.20 dL/g) waspurchased from DURECT Corporation (Pelham, Ala.). The MicroBicinchoninic Acid (BCA) Protein Assay Kit (23235) was purchased fromPierce (Rockford, Ill.). The Masson's trichrome kit was purchased fromAmerican MasterTech (Modesto, Calif.). The GDNF Emax Immuno-AssaySystems Kit was purchased from Promega (Madison, Wis.). Recombinanthuman Glial-Derived Neurotrophic factor (GDNF) produced in E. Coli waspurchased from Leinco Technologies (St. Louis, Mo.).

Fabrication of double-walled microspheres: To create double-walledmicrospheres, a 17.5% poly(lactic-co-glycolic acid) (PLGA) solution wascreated with 150 mg PLGA in dichloromethane. In a separate glassscintillation vial, a 10% solution of poly(lactide) (PLLA) of equalpolymer mass was prepared. After both polymers were fully dissolved,either 4 mg of FITC-BSA or 20 mg of lysozyme was added to the PLGAsolution and vortexed for ˜30 s to achieve a homogenous mixture. ThePLGA solution was then combined with the PLA solution and vortexed foran additional 60 s. This oil-in-oil emulsion was added drop-wise througha Pasteur pipette to 200 mL of aqueous 0.5% poly(vinyl alcohol) solutionstirring at 900 rpm for 3 h. Then, the polymer microspheres werecollected through centrifugation (1500 g for 10 min) and washed threetimes. Finally, the microspheres were lyophilized using a Labconcofreeze dry system (without a cryoprotectant) and stored in a desiccantat ˜20° C.

To encapsulate glial cell line-derived neurotrophic factor (GDNF), asolution of 40 ml (0.1 mg/mL) of GDNF, 100 mg of docusate sodium saltand 7 mg human serum albumin was prepared in 0.5 mL sterile water overice (formulation adapted from [22]). After mixing well, the solution wasfrozen and lyophilized. The protein/surfactant mixture was then added toPLGA already dissolved in dichloromethane and microspheres wereprepared, as described for lysozyme encapsulation.

GDNF release from double-walled microspheres: To determine the releasekinetics of GDNF from the PLGA/PLA double-walled microspheres, 10 mg ofmicrospheres were placed into Eppendorf tubes and incubated in 1 mL PBSat 37° C. At specified time points, the microspheres were vortexed,centrifuged for 10 min at 1500 g and the supernatant was replaced withfresh PBS. The amount of soluble GDNF in the collected samples wasanalyzed using an enzyme linked immunosorbent assay (ELISA)manufacturer's instructions. The optical density was recorded at 450 nmin an ELISA plate reader (Tecan, NC). The GDNF concentrations werecalculated against a 6-point standard curve, then adjusted to picogramsof GDNF per milligram of microspheres.

Fabrication of poly(caprolactone) disks and nerve guides:Poly(caprolactone) (PCL) disks were prepared to determine the effect ofnerve guide macrostructure on lysozyme release from the microspheres.Briefly, 15 mg of microspheres were added to a circular well (diameter=1cm, depth=0.5 cm) of a custom made silicone mold (FIG. 1A). Porous diskswere created by dissolving 1.35 g PCL in 15 mL ethyl acetate. To thedissolved polymer solution, sodium chloride impregnation wasaccomplished by adding NaCl in a 80% (v/v) amount. 200 ml of the polymerslurry was added to each mold and mixed well to distribute themicrospheres within the disk space. The ethyl acetate was allowed toevaporate and the sodium chloride was leached with distilled water.

PCL nerve guides were fabricated using a modification of previouslyreported methods [23]. Glass capillary mandrels 1.5 mm in diameter werecoated with a 17% w/v % aqueous solution of poly(vinyl alcohol) (PVA),air dried and then immersed into the polymer slurry (as described above)creating NaCl/PCL mandrel coatings. The ethyl acetate was allowed toevaporate for a minimum of 10 min between successive mandrel immersionsinto the polymer slurry. After the completion of the dip-coatingprocess, the resulting polymer conduits were submerged in distilledwater to allow for salt and PVA dissolution, and the guides were removedfrom the glass mandrels. The final wall thickness after 6 successiveimmersions of the mandrels into the polymer solutions was 600-700 mms.

To incorporate double-walled microspheres into the inner half of thenerve guide wall (FIG. 1B), 15 mg of microspheres were evenly spreadonto a drawn grid on parchment paper (FIG. 1C). After the firstimmersion of the glass mandrel into the PCL slurry, the ethyl acetatewas allowed to evaporate for only 30 s leaving a semihardened polymerlayer on the mandrel. This was then smoothly rolled across themicrospheres on parchment paper. The PCL with embedded microspheres wasallowed to dry for 10 min and then repeatedly coated with additionallayers of polymer as done in nerve guides without microspheres.

Evaluation of sodium chloride leaching from nerve guide walls: Todetermine the minimum amount of time required to leach the sodiumchloride from the nerve guide walls, nerve guides were fabricated asdescribed above (n=5). After ethyl acetate evaporation overnight, eachmandrel with the attached polymer guide was weighed. The mandrels werethen immersed in distilled water in 1 h increments. The dry weight ofeach guide was recorded between successive salt leaching periods.Previous work has indicated that in an 80% porous nerve guide, very fewclosed pores exist from which remaining salt crystals could not dissolve[23].

Visualization of microsphere distribution within nerve guides: To assessthe distribution of microspheres within PCL nerve guide walls, nerveguides with embedded microspheres encapsulating FITC-BSA were submergedin Optimal Cutting Temperature (O.C.T.) compound and frozen forsectioning with a cryostat. The nerve guides were then sectioned (15 mm)and either fixed onto glass slides or mounted on metal stubs usingdouble-sided copper tape. The overall location of microspheres withinnerve guides was then evaluated using low magnification (10×)fluorescent microscopy. To determine the effect of the nerve guidefabrication process on microsphere morphology, high magnificationscanning electron microscopy was performed. Transverse sections of nerveguides mounted on metal stubs were coated with gold using a Cressington108 Auto (Cressington,Watford UK) and then viewed with a JEM-6330F(JEOL, Peabody, Mass.) scanning electron microscope operating at 5 kVacceleration.

In vitro release of lysozyme from poly(caprolactone) disks and nerveguides: To determine the effect of the nerve guide material on lysozymerelease from double-walled microspheres, protein release kinetics werecompared between a known weight of microspheres embedded in PCL disks toan equal weight of free microspheres in solution. To accomplish this, 15mg of microspheres were first immersed in water for a period identicalto that used to remove the salt particulates from the PCL disks. Themicrospheres were then collected and incubated in 1 mL PBS at 37° C.Following incubation, the microspheres were centrifuged for 10 min at1500 g and the supernatant was removed. Five PCL disks prepared asdescribed above were individually placed into wells of a 48 well plate.To each disk, 0.6 mL of PBS solution was added and the well plate wasincubated for equal time periods as microsphere samples. To collect thereleasate, the disks were removed using sterile forceps and transferredinto clean wells where the PBS was refreshed. To measure the release oflysozyme from double-walled microspheres in PCL nerve guides, nerveguides were fabricated as described in Section 4.2. After the guideswere immersed in distilled water for 5 h, the guides were cut to 2 cmlengths and added to a clean Eppendorf tube. To each tube, 0.6 mL of PBSwas added and the guides were incubated at 37° C. for specified timeincrements. To collect the releasate, the guides were removed from eachEppendorf tube using clean forceps and added to new tubes in which thePBS was refreshed.

For measuring the lysozyme content of the releasate, a micro BCA proteinanalysis assay was perforated. Lysozyme standard or sample solution (1mL) and 1 mL of the Micro BCA working reagent were combined in a testtube and mixed well. All of the standard lysozyme samples as well as therelease samples were incubated simultaneously in a water bath for 1 h at60° C. After this period, the protein solutions were cooled to roomtemperature and 200 mL from each sample were transferred to a 48 wellplate and read with a plate reader at 562 nm (Tecan Spectraflour, NC).

Surgical methods: Following the guidelines of the University ofPittsburgh Institutional of Animal Care and Use Committee, 6 male Lewisrats (250-300 g, Harlan Labs) were used to evaluate the initial efficacyof GDNF released from PCL nerve guides for improved nerve regeneration.To implant the guide, each rat was anesthetized with an intraperitonealinjection of sodium pentobarbital (50 mg/kg). The sciatic nerve was thenexposed with a muscle splitting incision of the gluteal muscle. Thenerve was sharply transected ˜0.5 cm from the proximal bifurcation and0.5 cm of tissue was excised. After the proximal and distal nerve stumpswere allowed to retract, the exposed fascicles were trimmed and suturedwith 10-0 prolene epineurial mattress stitch 1 mm into each end of a 1.7cm nerve guide, creating a 1.5 cm defect (FIG. 2A). The gluteal muscleand skin were then closed with 4-0 vicryl suture. The animals wererandomly divided evenly amongst two groups, one of which receivedconduits with microspheres encapsulating GDNF and the other groupreceived identical conduits with empty microspheres as a negativecontrol.

Histological analysis: After 6 weeks, the animals were sacrificed withan overdose of sodium pentobarbital and the implanted guides wereharvested and immediately fixed in 4% paraformaldehyde. After the tissuewas fixed for at least 24 h, the nerve samples were washed with PBS andfixed in 1% osmium tetroxide for at least 2 h. After the nerve specimenswere dehydrated with increasing concentrations of ethanol (30-100%), thenerves were sectioned with a sharp razor blade at the proximal nervestump (PS), the proximal (PG), middle (MG) and distal (DG) regions ofthe nerve conduit, and at the distal nerve stump (DS) (FIG. 2). Thesections were then embedded in paraffin in descending order (FIG. 2C)and sectioned at 3 mm in thickness.

Masson's trichrome: For analysis of cellular and tissue infiltration ofthe nerve conduits, nerve sections from the negative control andexperimental animals were stained for Masson's Trichrome. Sectionednerves were first deparaffinized with xylene and rehydrated withdecreasing percent alcohol solutions (100% followed by 90% and then DIwater). Solutions from a Masson's trichrome kit were then used accordingto the protocol published by Di Scipio et al. [24].

Immunohistochemistry: For fluorescent visualization of Schwann cells,immunohistochemistry was performed on explanted nerve samples. Paraffinembedded specimens fixed in osmium tetroxide were first deparaffinizedas described above and then etched with H₂O₂ for 10 min as described in[24]. The samples were then blocked with 5% FBS with 0.02% triton-X inPBS for 1 h at room temperature. Antibodies against S-100 protein werethen added overnight at 4° C. (1:400 in 2.5% FBS and 0.02% triton-X inPBS). The samples were then washed three times with PBS and thesecondary antibody was added for 1 h at room temperature (1:1000 in 2.5%FBS and 0.02% triton-X in PBS). The samples were then washed thriceagain and the nuclei were detected using DAPI (0.6 mg/mL). The slideswere then mounted with a fluorescent mounting media.

Statistical analysis: A minimum repetition value of five was used whenmeasuring lysozyme release from PCL disks and nerve guides. Results areexpressed as the mean±standard deviation. Analysis of variance (ANOVA)was used to deteiinine statistical significance between experimentalgroups. The least significant difference method was used for multiplecomparisons with p<0.05.

6.2 RESULTS

Growth factor release from double-walled microspheres: Microspheres wereprepared encapsulating fluorescently labeled BSA for proteinvisualization, lysozyme to characterize protein release frommicrospheres embedded in PCL, and a neurotrophic factor: GDNF. Themicrospheres batch yield was 54.5%. GDNF release from double walledmicrospheres was determined using ELISA. The release profile indicatesan initial burst release of GDNF during the first day of microsphereincubation, with 4.8±0.4 ng GDNF measured per mg microspheres (FIG. 3A).Following the initial burst, GDNF was released with near zero-orderkinetics and by day 64 had a cumulative release of 6.4±0.02 ng GDNF permg microspheres. The overall percent bioactivity of GDNF released frommicrospheres alone was not assessed in vitro. Instead, GDNF integrity(bioactivity) was evaluated from the overall nerve guide delivery systemin vivo. Because we incorporated 15 mg of microspheres into each guide,we approximate the GDNF dosage during in vivo studies as ˜95 ng peranimal. The cumulative release of the GDNF (pg) per individual nerveguide (1.7 cm in length) is depicted in FIG. 3C, with values expressedas mean±std. dev. (n=4).

Minimization of salt leaching period: Leaching of sodium chloride fromthe nerve guide walls also results in a loss of protein from themicrospheres. Therefore, it is desirable to minimize the immersion ofnerve guides in water. Following fabrication of PCL nerve guidesimpregnated sodium chloride, the PCL guides were immersed in distilledwater for 1 h increments. FIG. 3B indicates that the majority (97%) ofNaCl was removed from the nerve guide wall after 1 h of immersion inwater. Beyond 5 h, no amount of measurable NaCl was further removed.

To validate the final PCL nerve guide weight, the guides were weighedafter 60 h of immersion in water and the final weight was unchanged.Because the initial weight of NaCl within nerve guides cannot beaccurately measured (guides are made by dipping mandrels in a largevolume of polymer/salt slurry), it is assumed here that only anegligible amount of NaCl remains within closed pores following theextended period of mandrel immersion in water. This assumption issupported by the very low frequency of closed pores detected in 80%porous PCL nerve guides [23].

Incorporation of double-walled microspheres into nerve guides: A novelrolling technique was used to embed double-walled microspheres into PCLnerve guides for sustained protein release. Fluorescently labeled BSAwas encapsulated in the double-walled microsphere to visualizemicrosphere distribution within the nerve guide wall. Fluorescentmicroscopy images were then taken of the microspheres and overlaid withlight microscopy images of the polymer nerve guide. As seen in FIG. 4A,by smoothly rolling the nerve guide mandrel across the microspheres andperforming several consecutive immersions of the mandrel into thepolymer solution, a nerve guide was created with microspheresdistributed evenly along the luminal wall of the nerve guide. BecausePLA is not soluble in ethyl acetate, the rounded morphology of themicrospheres was maintained following nerve guide fabrication (FIG. 4B).

Lysozyme release from double-walled microspheres embedded inpoly(caprolactone): A known weight of double-walled microspheres wasincorporated into PCL disks to measure the effects of nerve guidemacrostructure on protein release. Lysozyme was released frommicrospheres in a significantly higher concentration than frommicrospheres embedded in PCL (p<0.05, n=5). The cumulative amount ofprotein released at day 35 was not significantly different between freefloating microspheres and microspheres in disks (p>0.05) (FIG. 5A). Toanalyze the long-term release of protein from PCL nerve guides, 15 mg ofdouble-walled microspheres identical to those used in the disks wereembedded into the walls of 5 individually prepared nerve guides. Thelysozyme release pattern seen from nerve guides was similar to that seenfrom microspheres alone (FIG. 5B). There was a gradual, sustainedrelease of lysozyme until day 36 when ˜45% of the total lysozyme wasreleased. After this period, there was an increase in release rate andby day 49, 90% of lysozyme was released. No detectable amount oflysozyme was measured beyond day 56. As a BCA assay was used to detectlysozyme in solution, even partially degraded protein fragments wouldstill be detected. Therefore, the remaining 10% of encapsulated lysozymehad most likely adsorbed onto the remaining polymer matrix ofmicrospheres.

Masson's trichrome: PCL nerve guides with double-walled microsphereswere implanted across a 1.5 cm defect in the sciatic nerve to determinethe initial effects of GDNF delivery on nerve regeneration. The presenceof cellular infiltration, tissue formation and collagen content withinthe implanted conduits was visualized through Masson's trichrome stain.At low magnification (FIG. 6A), transverse images of proximal segmentsof PCL conduits implanted without encapsulated GDNF showed a thincollagen capsule surrounding the nerve guide (arrow). Within the lumenof the guide, there is incomplete tissue integration and a lack ofintercellular fibers. High magnification images (FIG. 6B) reveal thepresence of blood vessels (arrow head) and localization of cells nearthe inner surface of the nerve guide. There does not appear to be ameasurable number of nerve fibers present and the majority ofinfiltrating cells, such as fibroblasts and macrophages, are organizedaround the nerve guide wall and surround the microspheres (MS). Neitherthe experimental nor control guide appear to illicit a stronginflammatory response.

Visualization of nerve guides implanted with double-walled microspheresreleasing GDNF shows a higher concentration of intercellular fibers andtissue formation at the proximal segment of the guide (FIG. 6C). Thoughconnective tissue does not appear to be organized, high magnificationimages of the center of the nerve guide reveal the presence of cellsthroughout the entire interior of the guide (FIG. 6D). The presence of ablood vessel located near a microsphere in the nerve guide wall isindicated with an arrow head. In the distal region of the implantednerve guides, the empty conduit shows very little tissue formation inthe lumen of the guide in comparison to guides releasing GDNF (FIGS. 7Aand B). Analysis of high magnification light micrograph images confirmedthat in negative control guides, there is an absence of intercellularfibers and cells in the lumen of the guide (FIG. 7C). In GDNF conduits(FIG. 7D), tissue infiltration is not as developed as was seen in thesame proximal nerve segment, however there was an increase in collagencontent and intercellular fibers above the negative controls.

Immunohistochemistry: Results from immunohistochemical analysis of nervesections treated with anti-S100 antibodies showed that Schwann cellswere present in the proximal nerve guide segment in both negativecontrol and nerves that received guides with GDNF microspheres. However,as seen in FIG. 8A, the distribution of Schwann cells in control guideswas largely restricted to the middle of the nerve guide. Within thelumen of nerve guides releasing GDNF, Schwann cells are evident not onlyin the center of the conduit, but also surrounding the microspheres(FIG. 8B). At higher magnification, Schwann cells (red) are visualizedsurrounding the double-walled microspheres releasing GDNF (FIG. 8D) butare not present around non-encapsulating control microspheres (FIG. 8C).Analysis of the distal regions of the explanted nerve guide reveals thepresence of Schwann cells in the lumen of only those guides releasingGDNF. In FIG. 9A, DAPI staining of nuclei reveals the presence ofunspecified cells in smaller number in control guides as compared toGDNF guides (FIG. 9B). Also evident in FIG. 9B is a small population ofSchwann cells is present in the experimental guides not seen in thenegative control guides.

6.3 DISCUSSION

Direct bolus application of neurotrophic factors is not a suitablemethod for determining the long term effect of such molecules on nerveregeneration due to the short half life of growth factors in vivo.Therefore, it is desirable to use a controlled growth factor deliverysystem that is capable of delivering neurotrophins to Schwann cells andnerve stumps at the site of ligation in the sciatic nerve over asustained period. To accomplish this, we looked toward traditionalmicrosphere drug delivery systems as a method of encapsulating aneurotrophic factor. Typical microsphere release kinetics include aninitial burst of protein release within the first 24 h of placement inaqueous solution either in vitro or in vivo. To increase the time overwhich protein is released, PLGA microspheres were coated with anadditional layer of polymer (e.g. PLA), creating a double-walledmicrosphere. With this technique, the encapsulated growth factor isprotected in the core of the two-layered core-shell microspherestructure. Furthermore, by localizing a growth factor to the core of thedouble-walled microsphere, the amount of material through which theprotein must diffuse through is increased thus slowing the proteinrelease rate. In addition, double-walled microspheres with a core ofPLGA and a poly(L-lactide) (PLA) shell is an effective method ofincorporating the microspheres into PCL nerve guide walls. PLA is notsoluble in ethyl acetate, the solvent used to create the PCL/NaCl slurryfor nerve guide fabrication and therefore protects the microspheres fromdissolution during nerve guide fabrication.

In the present study, it has been demonstrated that GDNF can beencapsulated at a concentration measuring several nanograms permilligram of double-walled microspheres. The microspheres werereproducibly incorporated into PCL nerve guides in a distribution thatwas tailored to manufacturer specifications. Lysozyme release studiesfrom microspheres embedded in PCL disks suggest that the nerve guidematerial delays the initial release of protein into the aqueousenvironment, presumably because the protein is trapped within the porousarchitecture of the nerve guide wall. In the creation of our PCL nerveguides, it was initially hypothesized that this effect of nerve guidemacrostructure on the initial protein release kinetics would be ofbenefit for our application, as the initial burst of GDNF from themicrospheres (FIG. 3A) would be delayed and closer to zero-order.However, lysozyme release from the double-walled microspheres withinnerve guide walls was no longer detectable after 54 days while previouswork in our lab has shown that microspheres suspended in PBS releaseprotein for >80 days [21]. It is possible that released protein adsorbsto the PCL surface upon release and is entrapped within the nerve guidewall. Initial exposure of the PCL guide surface to blood proteinsfollowing implantation in vivo would likely minimize GDNF adsorptionupon release from microspheres.

While the benefits of many different neurotrophic factors have beenassessed in sciatic nerve defects (e.g. NGF, BDNF, CNTF) we chose toinitially implant nerve guides releasing GDNF because of the promisingresults described in literature. GDNF is a neuroprotective growth factorsecreted by Schwann cells in distal segments of peripheral nervesfollowing injury [25]. In addition, GDNF has been shown to preventavulsion-induced motor neuron death following complete nerve transectionwhereas nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), and insulin-like growth factor (IGF) all failed to enhance cellsurvival or cell size [26]. Finally, GDNF treatment with guidancechannels following spinal cord injury resulted in a reduction ofreactive astrocytosis and macrophage accumulation [27]. The efficacy ofGDNF toward improving peripheral nerve regeneration through nerveguidance conduits has also been evaluated in vivo. In a 2007 study byPatel et al., chitosan scaffolds blended with laminin-1 and 5 mg GDNFwere implanted across a 10 mm gap in the Lewis rat sciatic nerve [28].After 12 weeks, the chitosan/GDNF guides resulted in decreasedgastrocnemius muscle atrophy and restoration of functional strength thatwas comparable to autograft controls. Behavioral testing indicated GDNFtreatment groups regained sensation and improved gait kinematics. Inaddition, silicone conduits filled with GDNF gene modified Schwann cellshave resulted in significantly improved nerve conduction velocity,number and density of regenerated nerves, and the thickness of themyelinsheath of regenerated nerves than that seen in controls across a 10 mmdefect in the Wistar rat [29]. Finally, nerve guides with a GDNFreleasing rod increased the number of myelinated axons and the overallnumber of regenerated axons was four-fold higher than nerve guides withNGF [7].

Within published literature concerning growth factor delivery from nerveguides, there is a wide range of target growth factor dosage. Thisvariability could reflect the particular therapeutic index, half-life,or delivery method for each growth factor. For example, Lewin et al.,investigated the effect of brain-derived neurotrophic factor (BDNF) andCiliary neurotrophic factor (CNTF) on peripheral nerve transectionsthrough the delivery of 300 mg of each growth factor via osmotic pump(delivering approximately 9 mg/day) [18]. Lower dosages of nerve growthfactor (NGF) have been used within heparin-containing silicone nerveguides, with tested dosages of 5, 20 or 50 ng NGF/mL [16]. Additionally,while in vitro models using dorsal root ganglion explants have been usedto predict optimal growth factor combinations for axonal outgrowth [30],it is difficult to predict optimal dosages for implantation consideringthe increased complexity of an in vivo environment. Therefore, we didnot intend to deliver an optimal concentration of GDNF within thispreliminary nerve guide delivery system feasibility study. Instead, weestablished the highest weight of microspheres which could be embeddedwithin the nerve guides reproducibly, or 15 mg microspheres per 1.7 mmlong nerve guide. Upon establishing the efficacy of this nerve guidemodel it will be possible to adjust microsphere formulations forimproved encapsulation efficiency or to vary the weight of microspheresembedded within the nerve guide walls for establishing an ideal dose ofgrowth factor.

Our in vitro release studies suggest that GDNF was released continuouslyduring the 6 week period of guide implantation across the sciatic nervelesion. While nerve gaps treated with empty microsphere conduitsresulted in incomplete fibrotic tissue formation and scatteredfibroblast—like cells in the center of proximal segment of the excisednerve guides, nerve guides releasing growth factor reveal an increase incellular infiltration and tissue integration within the lumen of theconduits. In addition, tissue integration in the distal segments ofnerve guides was markedly improved in guides releasing GDNF. Highmagnification micrographs of the central portion of transverse segments(FIGS. 7B and C) reveal an overall increase in cellular infiltration andcollagen content following GDNF treatment. This increase in collagencontent could potentially supply an improved scaffold for Schwann cellmigration and support axonal outgrowth.

Detection of Schwann cell localization with immunofluorescence indicatedthat Schwann cells were present in proximal segments of PCL nerveguides. Fluorescent micrographs of control guides show a significantnumber of Schwann cells within the lumen of the guide (FIG. 8A) andSchwann cells do not appear to localize to the conduit wall ormicrospheres (FIG. 8C). Nuclear staining (DAPI) within the conduitcenter reveal a large presence of additional cell populations which mostlikely include fibroblasts and macrophages but were not positive forantibodies against neurofilament antibodies (FIG. 8C). However,micrographs of nerve guides with GDNF microspheres show a population ofSchwann cells encircling the microspheres, an indication of targetedmigration of Schwann cells toward a source of GDNF (FIGS. 8B and D).This result is significant for two reasons. First, a cellular responseto the encapsulated growth factor suggests that the released GDNF isbioactive and has not been completely denatured through the nerve guidefabrication process. Second, the migration of Schwann cells, presumablyfrom the lumen of the conduit, indicates that GDNF is being delivered tothe lumen of the nerve guide in a physiologically relevant concentrationand is not entirely entrapped within the porous nerve guide structure.

Nerve guides encapsulating GDNF also resulted in the presence of Schwanncells at the distal portion of transverse sections of explanted tissuethat was not seen in control guides (FIGS. 9A and B). Fluorescentmicrographs of the central portion of the nerve guide reveal an increasein both cellular infiltration (DAPI) and cells positive for antibodiesagainst S-100 protein in experimental guides while there were nodetectable positive for S-100 in control guides. The presence of Schwanncells in the distal segment of experimental nerve guides could be aresult of either Schwann cell proliferation or migration. Though GDNF istypically utilized in nerve regeneration as trophic factor for nervefibers and as a promoter of axonal growth [31] and branching [32,33],GDNF also causes a marked proliferation and migration of Schwann cells[33,34]. The effect of GDNF is RET (receptor tyrosine kinase)independent and instead, GDNF signaling mechanisms directly in Schwanncells through a GPI-anchored coreceptor (GFR-al) and neural celladhesion molecule (NCAM)[34]. Additionally, downstream signaling proteinkinases (PKA) participates in GDNF transcription, and therefore, GDNFpromotes a positive feedback loop for autocrine signaling in Schwanncells. This autocrine feedback loop has been reported to further enhanceSchwann cell migration and myelination [25], both of which are importantevents in nerve regeneration.

The presence of regenerated nerves into nerve stumps distal to theimplanted conduits was not assessed due to the early time point usedwithin this study. The intended purpose of this study was to establishthe feasibility of our overall nerve guide design for deliveringbioactive proteins to injured peripheral nerves from degradable polymerconduits. It was our hypothesis that an early time point would improvethe likelihood of observing a distinct cellular response within conduitswhich deliver growth factor and the negative control guides.Furthermore, because we did not anticipate nerve regeneration to havebeen completed across the large gap used within our animal model, we didnot look for functional recovery of lower leg muscle as they would notyet be properly innervated. Following the completion of this preliminarystudy, a longer time point will be used in future studies which aim todetermine the efficacy of our selected growth factor, GDNF, forimproving nerve regeneration.

Double-walled microspheres were fabricated as a method of encapsulatingGDNF, and subsequently incorporated into biodegradable polymeric nerveguides. Initial in vitro release studies from microspheres reveal asustained release of GDNF to day 50, a time point beyond our 6 weekimplantation period. An analysis of the effects of the nerve guidematerial, PCL, on release kinetics from incorporated microspheresrevealed that porous PCL delayed the initial period of protein releaseuntil day 35, when no significant difference in cumulative proteinrelease was measured. The polymer orientation of double-walledmicrospheres protected the microsphere morphology during PCL nerve guidefabrication. The microsphere core was composed of PLGA while the shellwas PLLA, a polymer that is insoluble in ethyl acetate which was used tocreate the PCL/NaCl slurry. Nerve guides embedded with microspheresencapsulated with FITC-BSA show a homogeneous distribution ofmicrospheres along the inner luminal circumference of the guide (FIG.4A) and SEM analysis indicate that the mechanical integrity of themicrospheres was maintained (FIG. 4B). Implantation of PCL nerve guideswith GDNF in a rat sciatic nerve defect resulted in an increase intissue integration in both the proximal and distal segments of the lumenof the nerve guide and an increase in Schwann cells in the distal regionof the guide. Migration of Schwann cells toward double-walledmicrospheres indicates that bioactive GDNF was encapsulated anddelivered to the internal environment of the nerve guide. The nerveguides created within this experiment indicate potential for examiningthe effect of a variety of growth factors within long gap peripheralnerve defects.

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[24] Di Scipio F, Raimondo S, Tos P, Geuna S. A simple protocol forparaffin-embedded myelin sheath staining with osmium tetroxide for lightmicroscope observation. Microsc Res Tech 2008; 71(7):497-502.

[25] Iwase T, Jung C G, Bae H, Zhang M, Soliven B. Glial cellline-derived neurotrophic factor-induced signaling in Schwann cells. JNeurochem 2005; 94(6):1488-99.

[26] Li L, Wu W, Lin L F, Lei M, Oppenheim R W, Houenou L. Rescue ofadult mouse motoneurons from injury-induced cell death by glial cellline-derived neurotrophic factor. Proc Natl Acad Sci USA 1995;92(21):9771-5.

[27] Iannotti C, Li H, Yan P, Lu X, Wirthlin L, Xu X-M. Glial cellline-derived neurotrophic factor-enriched bridging transplants promotepropriospinal axonal regeneration and enhance myelination after spinalcord injury. Exp Neurol 2003; 183(2):379-93.

[28] Patel M, Mao L, Wu B, VandeVord P J. GDNF-chitosan blended nerveguides:a functional study. J Tissue Eng Regen Med 2007; 1(5):360-7.

[29] Li Q, Ping P, Jiang H, Liu K. Nerve conduit filled with GDNFgene-modified schwann cells enhances regeneration of the peripheralnerve. Microsurgery 2006; 26(2):116-21.

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[31] Paratcha G, Ledda F. GDNF and GFR[alpha]: a versatile molecularcomplex for developing neurons. Trends Neurosci 2008; 31(8):384-91.

[32] Gordon T. The role of neurotrophic factors in nerve regeneration.Neurosurg Focus 2009; 26(2):E3.

[33] Hoke A, Ho T, Crawford T O, LeBel C, Hilt D, Griffin J W. Glialcell line-derived neurotrophic factor alters axon schwann cell units andpromotes myelination in unmyelinated nerve fibers. J Neurosci 2003;23(2):561-7.

[34] Paratcha G, Ledda F, Ibanez C F. The neural cell adhesion moleculeNCAM Is an alternative signaling receptor for GDNF family ligands. Cell2003; 113(7): 867-79.

7 EXAMPLE Sustained Growth Factor Delivery Promotes Axonal Regenerationin Long Gap Peripheral Nerve Repair 7.1 MATERIALS AND METHODS

Reagents: All chemicals were analytical grade or purer and werepurchased from commercial suppliers. Poly(vinyl alcohol) (PVA; averageMw9000-10,000, 80% hydrolyzed), poly(DL-lactide-coglycolide)(lactide:glycolide (50:50), Mw 40,000-75,000 units),PCL (Mw 65,000),dichloromethane, ethyl acetate, xylene, monoclonal anti-S100 andanti-Neurofilament, and phosphatebuffered saline (PBS) were allpurchased from Sigma-Aldrich.Poly-L-lactide (0.90-1.20 dL/g) waspurchased from Durect Corporation (Pelham, Ala.). The Masson's trichromekit was purchased from American MasterTech. The GDNF Emax ImmunoAssaySystems Kit was purchased from Promega. Recombinant human GDNF producedin Escherichia coli was purchased from Leinco Technologies.

Fabrication of nerve guides with double-walled microspheres:Double-walled microspheres were prepared as previously described(11,12). Briefly, a 17.5% poly(lactic-co-glycolic acid) (PLGA) solutionwas created with 150 mg PLGA in dichloromethane. In a separate glassscintillation vial, a 10% solution of poly(lactide) of equal polymermass was prepared. A solution of 40 μL (0.1 mg/mL) of GDNF and humanserum albumin in a 1:10 molar ratio was prepared in 0.5 mL sterile waterover ice (formulation adapted from ref.13). After mixing well, thesolution was frozen and lyophilized. The protein/surfactant mixture wasthen added to PLGA already dissolved in dichloromethane and vortexed for*30 s to achieve a homogenous mixture. The PLGA solution was combinedwith the poly(lactic acid) (PLA) solution and vortexed for an additional60 s. This oil-in-oil emulsion was added drop-wise through a Pasteurpipette to 200 mL of aqueous 0.5% PVA solution stirring at 900 rpm for 3h. Then, the polymer microspheres were collected through centrifugation(1500 g for 10 min) and washed three times. Finally, the microsphereswere lyophilized using a Labconco freeze dry system (without acryoprotectant) and stored in a desiccant at −20° C. Twenty PCL nerveguides were fabricated using a modification of previously reportedmethods (14). Glass capillary mandrels were coated with a 17% w/v %aqueous solution of PVA, air-dried, and then immersed into the polymerslurry creating NaCl/PCL mandrel coatings. Microspheres (15 mg) wereevenly spread onto a drawn grid on parchment paper (illustrated inref.11). After the first immersion of the glass mandrel into the PCLslurry, the ethyl acetate was allowed to evaporate for 30 s leaving asemi-hardened polymer layer on the mandrel. This was then smoothlyrolled across the microspheres on parchment paper. The PCL with embeddedmicrospheres was allowed to dry for 10 min and then repeatedly coatedwith additional layers of polymer as done in nerve guides withoutmicrospheres.

In vitro release of GDNF from nerve guides with double-walledmicrospheres: After fabrication, the guide was allowed to dry completelyand NaCl leaching was performed by immersing the guides in steriledeionized (DI) water for 5 h. Nerve guides were removed from glassmandrels used during initial preparation and cut to 1.7-cm lengths. Fromthis group of nerve guides, four were randomly selected for measuring invitro GDNF release kinetics. The remaining 16 were implanted into theexperimental group of animals. Long-term release studies were performedto approximate the amount of GDNF released during the duration of the invivo studies. Nerve guides were individually added to clean eppendorftubes. To each tube, 0.6 mL of PBS was added and the guides wereincubated at 37° C. for specified time increments. To collect thereleasate, the guides were removed from each eppendorf tube using cleanforceps and added to clean tubes with fresh PBS. The amount of solubleGDNF from the collected samples was analyzed using an enzyme linkedimmunosorbent assay (ELISA) per manufacturer's instructions. The opticaldensity was recorded at 450 nm in an ELISA plate reader (Tecan). TheGDNF concentrations were calculated against a six-point standard curve,and then adjusted to picograms of GDNF per milligram of microspheres.

Surgical methods: Following the guidelines of the University ofPittsburgh Institutional of Animal Care and Use Committee, 48 male.Lewis rats (250-300 g, Harlan Labs) were used to evaluate the initialefficacy of GDNF released from PCL nerve guides for improved nerveregeneration. Each rat was anesthetized with an intraperitonealinjection of sodium pentobarbital (50 mg/kg). The sciatic nerve wasexposed with a muscle splitting incision of the gluteal muscle. Thenerve was sharply transected *0.5 cm from the proximal bifurcation and0.5 cm of tissue was excised. After the proximal and distal nerve stumpswere allowed to retract, the exposed fascicles were trimmed and suturedwith 10-0 prolene epineurial mattress stitch 1 mm into each end of a1.7-cm nerve guide, creating a 1.5-cm defect. The gluteal muscle andskin were then closed with 4-0 vicryl suture. The animals were randomlydivided evenly amongst three groups, one of which received conduits withmicrospheres encapsulating GDNF and the other group received identicalconduits with empty microspheres as a negative control. The third groupof rats consisted of those which received a nerve isograft as a positivecontrol; that is, nerve grafts from sacrificial rats of the same strainwere used such that one donor animal produced two nerve grafts.

Functional assessment of nerve regeneration: Functional reinnervation ofthe lower limb muscles was assessed through three methods: video gaitkinematics, gastrocnemius muscle twitch force, and gastrocnemius wetweight.

Video gait kinematics: A high-speed (100 full frames per second) digitalvideo camera (Basler A6020 was used to record motion of the hindlimbduring walking along a 10-cm-wide runway (FIG. 10A). Black ink marks ofcontrast (3 mm diameter) or reflective markers (FIG. 10B) werepositioned over the iliac crest, greater trochanter, knee, ankle, andfifth metatarsal-phalangelal joints. During each test, the rat waspositioned at one end and encouraged to walk the length of the 60-cmrunway. This procedure is repeated until 15-20 steps were recorded withthe rat walking straight forward (FIG. 10C). FIG. 11 shows an imagetaken from the high-speed video of a rat walking on the runway. A customMatlab program was used to automatically track the location of each markof contrast in all frames of the video. The marker locations were thenused to construct line segments connecting each pair of adjacentmarkers. Intersegmental joint angles for the hip, knee, and ankle jointswere calculated using Equation 1, which results from the dot productrule.

0=A·B′/|A∥B|

In this equation, the vectors A and B denote the line segments proximaland distal to the joint. The intersegmental angle (y) was computed asthe inverse cosine of the dot product of vectors A and B, normalized bytheir respective lengths. The joint angle trajectories were computedfrom the measured marker positions. The foot touchdown and liftoff timeswere identified by visual inspection of the video and used to parse theswing (liftoff to touchdown) and stance (touchdown to liftoff) phases ofthe step cycle. The range of angular motion (maximum−minimum angles) foreach joint was calculated during the swing and stance phases. Thesemeasures were then used for comparing gait kinematics across time pointsand treatment groups.

Gastrocnemius twitch force: Immediately before sacrifice, animals wereanesthetized with sodium pentobarbitol and the injured sciatic nerve wasexposed with a muscle splitting incision made following the scar lineremaining from graft or guide implantation. After freeing the isograftor regenerated nerve from the superficial and underlying muscle tissues,a bipolar nerve cuff electrode (1.5 mm diameter, Microprobe, Cat #NC(1.5)24) was placed around the nerve. Because the implanted nerveconduit enclosed the regenerated nerve, the PCL material had to becarefully removed such that sufficient nerve material was available forcontact with the nerve cuff The lower gastrocnemius was then completelyisolated from the anterior and posterior tibialis muscles and theAchilles tendon was cut and fastened to a force transducer using atransfixation stitch and 4-0 silk sutures (FIG. 12). Finally, tocompletely stabilize the femur position, the animal's foot, knee, andback were immobilized on the data acquisition board using 16-0 gaugeneedles. Gastrocnemius twitch force was measured during stimulation ofthe sciatic nerve via the bipolar nerve cuff electrode. Tension in thesuture connecting the gastrocnemius muscle to the force transducer wasadjusted to obtain the maximum force. Stimulation pulses (200 ms wide;Model A320R; WPI) were applied at supramaximal intensity and theresulting twitch force was recorded at a sampling rate of 3 s (ModelUSB6009; National Instruments). The peak twitch force (relative tobaseline) that was measured for five consecutive stimulation pulses wasthen calculated and optimized for the strongest stimulation points ofcontact between the nerve cuff and the sciatic nerve.

Gastrocnemius muscle weight: After muscle force measurements, theanimals were euthanized with an overdose of sodium pentobarbital (100mg/kg intraperitoneal [IP]). A longitudinal incision in the lower legparallel to the extension of the Achilles tendon and gastrocnemius(gastroc) muscle was made followed by a dissection of the skin whichadequately exposed the gastroc muscle. The two proximal tendonousinsertions of the gastroc in the femoral area were identification andsectioned. The distal gastroc insertion in the heel through the Achillestendon was then severed for the extraction of the gastroc muscle. Themuscle was then immediately weighed. The wet muscle mass of theunoperated contralateral control was compared to the muscle mass of thegastroc from the injured leg.

Histological analysis: After the animals were sacrificed the implantedguides or isografts were harvested and immediately immersed in afixative. Nerves prepared for paraffin embedding for MTC stain and IHCwere fixed in 4% paraformaldehyde. Samples prepared for histomorphometryanalysis were fixed in 2.5% glutaraldehyde. After the tissue sampleswere treated with fixative for at least 24 h, the samples were washedwith PBS and postfixed in 1% osmium tetroxide for either 2 h in samplepreparation for MTC and IHC analysis or 24 h for those samples preparedfor histomorphometry. Nerve specimens were then dehydrated withincreasing concentrations of ethanol (30%-100%) and sectioned with asharp razor blade at the proximal nerve stump, the proximal, middle, anddistal regions of the nerve conduit, and at the distal nerve stump asdescribed previously (11). The sections were then embedded in paraffinor epoxy in descending order and sectioned at thicknesses of 3 μm (IHC,MTC) or 0.5 μm (histomorphometry).

Masson's trichrome: For analysis of cellular and tissue infiltration ofthe nerve conduits, including collagen formation, nerve sections fromthe negative control and experimental animals were stained for Masson'sTrichrome. Sectioned nerves were first deparaffinized with Xylene andrehydrated with decreasing percent alcohol solutions (100% followed by90% and then DI water). Solutions from a Masson's trichrome kit werethen used according to the protocol published by Di Scipio et al. (15).

Immunohistochemistry: For fluorescent observation of Schwann cells, IHCwas performed on explanted nerve samples. Paraffin-embedded specimensfixed in osmium tetroxide were first deparaffinized as described aboveand then etched with peroxide for 10 min as described in ref.15 Thesamples were then blocked with 5% fetal bovine serum (FBS) with 0.02%triton-X in PBS for 1 h at room temperature. Antibodies against S-100protein were then added overnight at 4° C. (1:400 in 2.5% FBS and 0.02%triton-X in PBS). The samples were then washed three times with PBS andthe secondary antibody was added for 1 h at room temperature (1:1000 in2.5% FBS and 0.02% triton-X in PBS). The samples were then washed thriceagain and the nuclei were detected using 4′,6-diamidino-2-phenylindole(0.6 μg/mL). The slides were then mounted with a fluorescent mountingmedia.

Histomorphometry: Nerve guides were fixed using 2.5% glutaraldehyde,embedded in epon, and cut into 0.5-μm cross sections using anultramicrotome (Reichert Ultracut). Sections were then mounted ontoglass slides and stained with 1% toluidine blue dye for imaging. AHitachi (model KP-M1AN) digitizing camera was mounted on a Zeiss PrimoStar microscope for image acquisition. A 100× oil immersion objectivelens was used to produce digital images at a final magnification of1000×, with a pixel size of 0.125 μm as calibrated with a stagemicrometer.

The Leco IA32 Image Analysis System (Leco) with custom calculationroutines (macros) was used as developed by Hunter et al.16 8-bitmonochrome images were acquired and thresholded for determining myelincomposition. As described by Hunter et al., manual adjustments were madeto photomicrographs displayed on an attached monitor such that debrisand nonviable nerve fibers were removed. Viable axons were defined asdark myelin rings enclosing clear fiber areas devoid of debris or cellnuclei. Myelin width, axon width, and fiber diameter were thenautomatically calculated through software analysis of red and greenbitplane identifiers. From these primary measurements, g-ratio (theratio of the axonal diameter divided by the diameter of the axon and itsmyelin sheath) and nerve fiber density (fiber number/mm2) werecalculated.

Statistical analysis: A minimum repetition value of four was used whenmeasuring GDNF release from nerve guides. Results are expressed as themean|standard deviation. Analysis of variance was used to determinestatistical significance between experimental groups. The leastsignificant difference method was used for multiple comparisons withp<0.05.

7.2 RESULTS

GDNF release from nerve guides with double-walled microspheres:Long-term release studies of GDNF from PCL nerve guides withdouble-walled microspheres were performed to approximate the releaseprofile of the growth factor in vivo. Nerve guides embedded withdouble-walled microspheres encapsulating GDNF were incubated in PBS andreleased growth factor was quantified using an ELISA system. As shown inFIG. 3C, the release of GDNF from the PCL nerve guides did not exhibitthe typical burst release profile seen in single-walled microspherestudies. At day 3, only *2.9% of the total released protein is liberatedinto solution. GDNF release is nearly linear until day 56, at whichpoint *89.0% of the total growth factor is released. After this point,GDNF release is consistent to day 112, when the 16 week in vivo studieswere complete. Assuming that the majority of microspheres weighed fornerve guide preparation were successfully embedded and secured into thenerve guide walls, the encapsulation efficiency for GDNF nerve guideproduction was ˜1%.

Implantation of PCL nerve guides: Upon exposure of the injured sciaticnerve at sacrifice, remaining PCL conduits were soft and pliable andboth sutures intact. Nerve guides were well vascularized with a softfibrous coating (FIG. 13A). The proximal and distal ends of the nerveguides were completely sealed with a fibrous capsule and small neuromaswere apparent at both guide ends. Before separating the nerve guide fromthe regenerated nerve, a pinch test was administered proximal to anyanastamosis to determine if a muscle reflex could be observed. In 75% ofthe animals treated with an empty PCL conduit (e.g., 12/16 rats), alower limb reflex was observed. However, often the regenerated nervethrough the negative control conduits was delicate, and attempts toremove the conduit for placement of a nerve cuff resulted in disruptionin nerve continuity. It was also noted that the regenerating nerve grewadjacent or into the porous walls of the PCL conduit. Nerve guidesimplanted with GDNF releasing microspheres were also observed as wellvascularized and sheathed in a soft fibrous coating. Results from apreliminary pinch test indicated that lower limb reinnervation was seenin 100% of the experimental animals. Furthermore, the nerves appeared togrow through the open lumen of the nerve guides and could be easilyseparated from the conduits for electrophysiology studies (FIG. 13B).

Functional assessment of nerve regeneration: Video gait kinematics: Gaitkinematics from animals receiving a PCL nerve guide without growthfactor (n ¼ 3) and animals which received GDNF releasing guides (n ¼ 16)after sciatic nerve injury are shown in FIG. 14. At the week 1 timepoint, all animals show a large reduction in the RoM at the ankle duringthe stance and swing phases; the stance-phase RoM for the knee was alsoreduced. Injured animals are unable to generate muscle force to extendthe knee, reaching only ˜90° of extension during stance (FIG. 14C)compared to 130° in the healthy animal. The ankle, which normally flexes˜70° to lift the foot during the swing phase, extends passively duringswing after injury (FIG. 14E). To compensate for reduced motion at theankle and knee, the hip joint shows a large increase in RoM as the hipis hyperflexed to provide sufficient ground clearance, compensating forthe reduced motion at the ankle and knee. By week 15, the stance phaseRoM for the ankle and knee return to baseline levels, and the RoMbetween experimental and negative control animals was not significantlydifferent between groups at any time point.

Functional assessment of nerve regeneration: Gastrocnemius twitch force:The mean measured gastroc twitch force was 0.59±0.28 N, 0.44±0.22 N, and0.07±0.07N for animals treated with an isograft (n=11), a nerve conduitwith GDNF (n=6) and animals treated with a PCL conduit and no growthfactors (n=7), respectively. The average twitch force between isograftanimals and those within the experimental GDNF group was notsignificantly different (p=0.1754), whereas both of these groups showedsignificantly improved twitch force above negative control animals (FIG.15).

Functional assessment of nerve regeneration: Gastrocnemius muscle wetweight: The wet weight of recovered gastroc muscles from injured legs asnormalized to contralateral uninjured controls are recorded in Table 1.The normalized values of muscles from animals treated with isografts asa positive control for nerve regeneration were statistically higher thanboth the experimental GDNF group as well as the PCL guides withoutgrowth factor delivery (p<0.01).

TABLE 1 RECORDED WET WEIGHTS OF INJURED GASTROCNEMIUS MUSCLE ASNORMALIZED TO CONTRALATERAL CONTROL Treatment description Gastrocnemiuswet weight (%) Isograft (n = 11) 46.6 ± 12.4^(a) PCL guide + GDNF (n =7) 24.8 ± 5.7 Empty PCL guide (n = 6) 21.7 ± 4.4 ^(a)Indicatesstatistical significance from experimental groups, p < 0.01. PCL,poly(caprolactone); GDNF, glial cell line-derived neurotrophic factor.

Histological analysis: Masson's trichrome: The regeneration of nerveacross a 1.5-cm defect was evaluated at 16 weeks. Low magnificationtransverse sections of the proximal portion of negative control conduitsreveal a high degree of collagen content within the lumen of the guide(blue) with tissue integration throughout the entirety of the guidelumen (FIG. 16A). Regenerated nerve is evident near the PCL nerve guidewall (black box) and, as evident at higher magnification, isdisorganized and sparse (FIG. 16B). Nerve tissue is also evident withinthe lumen of nerve guides releasing GDNF; however, fibers are located inthe center of the lumen surrounded by newly formed tissue (black circle,FIG. 16C). High-magnification light micrographs reveal a large number ofsmall nerve fibers that are well organized and thinly myelinated (FIG.16D).

Low-magnification brightfield images of transverse sections from controlPCL guides reveal that regenerated nerve tissue was not evident withinmid (FIG. 17A) or distal segments (FIG. 17B). Tissue integration appearsincomplete and few blood vessels are observed. However, in both the midand distal regions of conduits releasing GDNF, there was regeneratednerve tissue within the lumen of the guides. Low magnificationmicrographs of the midline of explanted conduits (FIG. 17C) showcollagen formation and tissue integration supporting the regeneratednerve fibers seen at higher magnification within FIG. 17D. Additionally,nerve tissue, including collagen and axons, is also evident withindistal regions of the GDNF releasing conduits within both low (FIG. 17E)and high magnification (FIG. 17F) micrographs.

Histological analysis: IHC: Nerve tissue was evident in the proximalregion of explanted conduits 16 weeks after sciatic nerve transectionand conduit implantation. Fluorescent images reveal the presence of bothnerve fibers (neurofilament proteins: green) and Schwann cells (S-100:red) in both negative control PCL guides and guides releasing GDNF.However, nerve tissue within control PCL guides (FIG. 18A) was localizedto the internal border of the nerve guide wall and appears disorganizedwhereas nerve tissue was centrally located and well organized withinGDNF releasing guides (FIG. 18B). Schwann cells and nerve fibers werenot detectable within the middle (FIG. 18C) or distal segments (FIG.18E) of control nerve guides, whereas robust Schwann cell populationswere observed across the entire length of GDNF releasing guides (FIGS.18D, F).

Histological analysis: G-ratio and nerve fiber density: Monochromeimages of the proximal nerve stump, proximal isograft or nerve guide,distal isograft or nerve guide, and distal nerve stump were acquired andthresholded to identify viable axons. Using semi-automated software,myelin width, axon width, and fiber diameter were calculated. From theseprimary measurements, g-ratio and nerve fiber density (fiber number/mm²)were determined. Within the isograft-positive control nerve samples, theg-ratio of axons was consistent within each section from the proximalnerve stump to the distal nerve stump with a measured range of 0.40 to0.42 (FIG. 19A). The g-ratio of nerve fibers within GDNF releasingguides were higher for all measured transverse sections, with averagevalues of 0.55, 0.59, 0.56, and 0.53 for the proximal nerve stump,proximal graft or guide, middle graft or guide, and distal nerve stumpsections, respectively, which are not statistically significantlydifferent from within measured groups. These measured values from GDNFguides approach an uninjured g-ratio range of 0.6-0.7(gray bar). Fibersfrom control PCL guides were observed to have a lower g-ratio valuewithin both the proximal nerve stump (0.47) and the proximal segment(0.45) of the explanted nerve guide. No fibers were evident in the midor distal regions of negative control PCL guides for measurementcalculations.

Evaluation of nerve fiber density throughout the length of explantednerve samples reveals an increased density of fibers per mm² for boththe GDNF and control nerve guides as compared to isograft positivecontrols, an indication of nerve regeneration (FIG. 19B). As seen infiber g-ratio results, isograft sections had a small range of fiberdensities (11,300-12,200) progressing from the proximal to the distalnerve stumps, indicating limited axonal sprouting occurred within thegraft The density of fibers within GDNF releasing guides was higher thannegative control guides for all measured segments and there were noviable nerve fibers in negative control nerve guides beyond the midregion of the explanted conduits.

7.3 DISCUSSION

The efficacy of GDNF toward improving peripheral nerve regenerationthrough nerve guidance conduits has been evaluated in vivo. Patel et al.reported chitosan scaffolds blended with laminin-1 and 5 μg GDNF wereimplanted across a 10-mm gap in the Lewis rat sciatic nerve (9). After12 weeks, the chitosan/GDNF guides resulted in decreased gastrocnemiusmuscle atrophy and restoration of functional strength that wascomparable to autograft controls. Behavioral testing indicated that GDNFtreatment groups regained sensation and improved gait kinematics. Inaddition, silicone conduits filled with GDNF gene-modified Schwann cellshave resulted in significantly improved nerve conduction velocity,number and density of regenerated nerves, and the thickness of themyelin sheath of regenerated nerves than that seen in controls across a10-mm defect in the Wistar rat (10). Finally, nerve guides with a GDNFreleasing rod increased the number of myelinated axons and the overallnumber of regenerated axons was four-fold higher than nerve guides withnerve growth factor (7). Experiments examining GDNF release from nerveguides for 6 weeks suggested that bioactive GDNF was delivered toendogenous cell populations and markedly improved cellular integrationand tissue formation across the nerve injury (11). Although nerve gapstreated with empty microsphere conduits resulted in incomplete fibrotictissue formation in the center of proximal segment of excised nerveguides, nerve guides releasing GDNF had an increase in cellularinfiltration and tissue integration within the lumen of the conduits.Additionally, we observed an increase in cellular infiltration andcollagen content after GDNF treatment, and we hypothesize that this leadto an improved scaffold for Schwann cell migration and supported axonaloutgrowth within this longer 16-week in vivo study. Within this study,we have shown that GDNF is released from nerve guides in vitro for over100 days. When implanted into rat sciatic nerve defects, a higherdensity of nerve tissue regenerated through the center of conduits asopposed to negative control guides (e.g., empty microsphere PCLconduits), which produced nerve tissue bolstered by the wall of thenerve guide. Regenerated nerve tissue from within GDNF releasing guidesappeared to have an increased level of collagen and intercellular tissueas observed with MTC. Quantification of high magnification lightmicrographs through software analysis supported visual impressions ofimproved nerve repair with GDNF treatment. Animals receiving GDNF had ag-ratio that approached native, uninjured levels, and were improvedabove both isografts and negative controls. Additionally, the mean nervefiber density of regenerated nerves was highest within conduitsreleasing GDNF, and was statistically higher than isograft controls atthe proximal end of conduits (p<0.05). Finally, immunofluorescentmicrographs identifying Schwann cells show a large population of Schwanncells throughout the length of conduits releasing GDNF, whereas very fewSchwann cells are detectable in the middle and distal segments ofnegative control guides. The presence of Schwann cells within the lumenof the guides is a positive indication for nerve repair, as severalstudies have shown that higher numbers of Schwann cells result in morerobust nerve regeneration (17-19). Analysis of muscle reinnervationthrough quantification techniques of rat gait proved challenging usingthe techniques described herein. While mass loss was statisticallyequivalent when comparing wet muscle weight in GDNF treated and emptynerve guides, muscle function through twitch force may be moreindicative of nerve regrowth and reinnervation of target end organs.This study suggests that measuring muscle twitch force is an improvedmethod of observing nerve regeneration to target muscles over thedescribed timepoint of 15 weeks. It is very possible that at a longertimepoint, the muscle wet weights between the control and experimentalgroups would have been different due to the continued lack of nerveinnervation in the gastrocnemius muscle of rats treated with empty nerveguides. No significant difference in joint angle RoM was measuredbetween the GDNF experimental group and the negative control. Thedevelopment of a technique for analyzing gait kinematics through videorecording was undertaken as a method of circumventing those challengesseen with sciatic functional index measurement (20). However, uniqueobstacles were presented within this method of gait analysis. First,animal behavior is varied, and often animals were observed as walkingwith a unique head position, speed, or step pattern. In addition,animals would traverse the walkway only a few times before losinginterest in the activity and could not be trained to proceed with asmooth gait pattern despite incentives provided by the operator. Becauseof this, step cycles were used for measuring joint angles from onlythose steps that were consecutive and were determined to be of a walkingnature (at least one foot always on the ground). The use of a treadmillwould greatly improve the consistency seen in joint angle measurementsand may allow this technique to be a more robust method for detectingearly improvements in gait kinematics after lower limb nerve injury.

Within this study, we have shown that GDNF can be successfullyencapsulated in double-walled microspheres and released in a controlledmanner from PCL nerve guides for over 100 days in vitro and improvesnerve regeneration in vivo. Upon the initial exposure of the site ofnerve injury, nerve tissue was observed exiting the distal end ofimplanted conduits in only 70% of the control PCL guides, whereas 100%of the animals implanted with GDNF releasing guides had nerve trunksthroughout the length of the conduits and integrating into the distaltarget muscles. In addition, the measured gastrocnemius twitch force, anindication of muscle atrophy and reinnervation, was significantlyimproved by a difference of sixfold in animals treated with GDNFreleasing conduits as opposed to those that received empty PCL conduits.Markedly, the twitch force between animals treated with isografts,considered to be the gold standard for nerve repair, was notsignificantly different from animals receiving GDNF releasing conduits.Although this study evaluated nerve repair after treatment with a singlegrowth factor, the nerve guide design is modular; therefore, these PCLconduits described herein lend themselves easily toward investigation ofa variety of different therapeutics for improved nerve regeneration.

7.4 REFERENCES

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The publication Biomaterials 31 (2010) p. 2313-2322, available online onDec. 7, 2010 is incorporated by reference in its entirety. Theabove-mentioned patents, applications, test methods, and publicationsare hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the fully intended scope of theappended claims.

1-10. (canceled)
 11. A method of making an implantable medical device comprising: (a) forming a first mixture of a first polymer and a first solvent; (b) forming a second mixture of a second polymer and a second solvent; (c) adding an active agent to the first mixture and/or the second mixture; (d) combining the first mixture and the second mixture to form a third mixture; (e) introducing the third mixture to a third solvent to form microspheres; (f) isolating the microspheres from the third solvent; and (g) introducing the isolated microspheres to a biodegradable polymer layer.
 12. The method of making the implantable medical device of claim 11, wherein the first polymer is poly(lactic-co-glycolic acid).
 13. The method of making the implantable medical device of claim 11, wherein the active agent is a neurotrophic factor.
 14. The method of making the implantable medical device of claim 13, wherein the neurotrophic factor is glial cell-line derived neurotrophic factor.
 15. The method of making the implantable medical device of claim 13, wherein the neurotrophic factor is glial growth factor
 2. 16. The method of making the implantable medical device of claim 11, wherein the second polymer is poly(lactide).
 17. The method of making the implantable medical device of claim 11, wherein the first mixture and the second mixture are combined by vortexing to form the third mixture, and the third mixture is added dropwise to the third solvent.
 18. The method of making the implantable medical device of claim 11, wherein the microspheres are isolated from the third solvent by centrifugation.
 19. The method of making the implantable medical device of claim 11, wherein the biodegradable polymer layer comprises poly(caprolactone).
 20. The method of making the implantable medical device of claim 11, wherein the double-walled microspheres are introduced to the biodegradable polymer layer via an electrospinning or a polymer casting technique. 21-27. (canceled) 